1) Field of the Invention
The present invention relates to novel gastric acid- and bile salt-resistant Lactobacillus isolates having the ability to lower and assimilate serum cholesterol and the to use thereof.
2) Description of the Related Art
“Lactic acid bacteria” are a group of bacteria that can ferment saccharides and that produce lactic acid as a major product. The generally accepted morphological and physiological characteristics of lactic acid bacteria reside in that they are: (1) Gram positive; (2) rod-shaped or disk-shaped; (3) catalase-negative; (4) capable of converting metabolized glucose into more than 50% of lactic acid; (5) nonsporeforming; (6) nonmotile; and (7) microaerobic.
Up to 1980, lactic acid bacteria were known to include, in general, four genera, namely, Lactobacillus, Streptococcus, Leuconostoc and Pediococcus (W. C. Frazier and D. C. Westhoff, 1978, Food Microbiology, 3rd ed. McGraw-Hill, Inc., New York, USA). In a broader sense, lactic acid bacteria further comprise two genera of Bifidobacterium and Sporolactobacillus. In recent years, microorganisms have been explicitly classified as a taxonomic group according to DNA homology and rDNA sequence comparison and analysis in the classification system, and have been given a position in taxonomy. To Applicants' knowledge, the family of lactic acid bacteria has expanded to include 16 genera and 223 species by December 1999.
The so-called “probiotics” are live bacteria of a single type or a mixture of bacteria of different types which, after being ingested by the human or animal host, can improve gastrointestinal microbial balance in the human or animal intestinal tract (O'sullivan et al. (1992), Trends in Food Sci. Technol., 3:309-314; Fuller, R., P. J. Heidt, V. Rush and D. van der Waaij. (eds.) (1995), Probiotics: prospects of use in opportunistic infections. Old Herborn University Seminar Monograph No. 8, pp. 1). Lactobacillus and Bifidobacterium are the most widely known and used probiotics.
In 1908, Dr. Eli Metchnikoff proposed the theory that the consumption of large amounts of yogurt containing strains of Lactobacillus sp. would result in the replacement of toxic-producing bacteria normally present in the intestine, and would thus result in longevity-without-aging (Eli Metchnikoff, 1908, The Prolongation Of Life, Ed. P. Chalmers Mitchell, G. P. Putnam's Sons, The Knickerbocker Press, New York & London). Recent studies and clinical tests have also shown that Lactobacillus is critically related to health. Therefore, Lactobacillus has received wide attention in recent years.
Lactic acid bacteria not only play an important role in the complex biosystem of the intestines, they also have probiotic effects on the host. The recognized effects of lactic acid bacteria include: improving nutritional value of host feed; promoting the synthesis of vitamins and the production of enzymes (J. Denter and B. Bisping, 1994, Int. J. Food Microbiol., 22:23-31), inhibiting the growth of intestinal pathogenic bacteria and maintaining the balance of normal intestinal microflora (Hose, H. and Sozzi, T. (1991), J. Chem. Technol. And Biotech. 51:540-544), producing antibodies to enhance immunity of the host (H. Majamaa et al. (1995), Journal of Pediatric Gastro-enterology and Nutrition, 20:333-338), reducing the risk of colon cancer, and suppressing tumor formation (E. J. Schiffrin et al., 1997, Am. J. Clin. Nutr., 66:515S-520S). Researches also show that consumption of milk fermented with Lactobacillus can lower serum cholesterol levels.
Cardiovascular disease has been indicated as a major leading cause of death in industrial countries. In the United States, more people died of coronary heart disease than those of cancer and other diseases, and about three-quarters of the deaths are caused by atherosclerosis and complications thereof. High serum cholesterol level is one of the causes of cardiovascular diseases and atherosclerosis (Kannel et al. (1979), Ann. Intern. Med., 90:85-91; Pekkanen et al. (1990), New England J. Med., 322: 1700-1707). In Taiwan, cerebrovascular and cardiovascular diseases are on the list of top ten leading causes of death, and are the major causes of illness and death in old people. Therefore, hypercholesterolemia is a cause of illness that should not be ignored.
In 1974, Mann and Spoerry found that serum cholesterol levels in men decreased after consumption of yogurt fermented with a Lactobacillus strain, whereas no significant change was observed in those that consumed fresh milk (Am. J. Clin. Nutr., 27:464-469, 1974), which spurred extensive researches on the application of lactic acid bacteria and their effects in lowering cholesterol.
K. K. Grunewald reported in J. of Food Science:47:2078-2079 (1982) that the serum cholesterol level in rats could be significantly decreased when the rats were given feeds containing 10% milk fermented by Lactobacillus acidophilus for four weeks. It was also reported by Danielson et al. in J. Anim. Sci.: 67:966-974 (1989) that acidophilus yogurt reduced serum cholesterol and low density lipoproteins (LDLs) in boars fed with a high-cholesterol diet in combination with yogurt fermented with Lactobacillus acidophilus LA16 isolates for a period of 56 days, but had no effect on serum triglycerides and high density lipoproteins (HDLs).
The aforesaid report, which is related to an isolate strain of Lactobacillus acidophilus, LA16, is one of the many works by the research group headed by Dr. Khem M. Shahani on the characteristics and bioactivities of Lactobacillus acidophilus. Specifically, Dr. Shahani's group had conducted fairly extensive researches on the characteristics and bioactivities of a specially isolated and cultured strain of Lactobacillus acidophilus DDS-1 of human origin, including studies on its effect in reducing serum cholesterol level. The DDS-1 isolated strain was later patented (information regarding DDS-1™ is available on the website of Nebraska Cultures, Inc.
In 1997, Akalin et al. compared the effects of ordinary yogurt (fermented by Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaticus) and acidophilus yogurt (fermented by Streptococcus thermophilus and Lactobacillus acidophilus) on serum cholesterol levels in mice, and found that the ability of acidophilus yogurt to reduce serum cholesterol concentrations was significantly higher than that of the ordinary yogurt (A. S. Akalin et. al. (1997), J. Dairy Sci., 80:2721-2725).
De Smet et al. reported in British J. of Nutrition, 79:185-194 (1998), that the fecal output of bile salts in pigs fed with Lactobacillus acidophilus for four weeks (from week 3 to week 7) increased significantly, and the total serum cholesterol concentrations in the pigs also decreased significantly. It was therefore concluded that the enzymatic activity of bile salt hydrase (BSH, E.C.3.5.1.24) within Lactobacillus might be the mechanism responsible for lowering serum cholesterol in the treated pigs.
Cholesterol in humans can be synthesized by the liver, and can also be ingested from meat. There are two pathways for the excretion of cholesterol: (1) forming cholic acid as a result of liver metabolism, which is then conjugated with glycine or taurine to become water-soluble and to subsequently form bile salts such as glycocholates or taurocholates, with potassium or sodium ions for excretion in feces; and (2) forming steroid hormones that are excreted in urine as a result of hormone metabolism; however, only a small portion of the cholesterol is excreted in this manner.
Bile salts are water-soluble final products in cholesterol metabolism. These salts may enter the enterohepatic circulation and, because of the enzymatic activity of bile salt hydrase (BSH) of enteric bacteria, including Lactobacillus, Enterococcus, Peptostreptococcus, Bifidobacterium, Clostridium and Bacteroid, etc., cholic acids can be separated from glycine or taurine to yield deconjugated bile salts. The deconjugated bile salts do not dissolve in water, and are likely to co-precipitate with serum cholesterol for excretion out of the body.
In addition to the aforesaid, it is reported that the metabolism mechanism of cholesterol includes both assimilation and co-precipitation.
In 1985, it was pointed out that Lactobacillus acidophilus could adhere to and assimilate cholesterol, and that, in a medium containing 0.3% oxgall, Lactobacillus acidophilus could achieve a higher cholesterol reduction (S. E. Gilliland et al. (1985), Appl. Environ. Microbiol, 49: 377-381). Likewise, it was reported by Noh et al. in J. Dairy Sci. (1997), 82: 3107-3113 that Lactobacillus acidophilus was able to incorporate cholesterol into cellular membranes, and the incorporated cholesterol could be further assimilated and metabolized to form substances required by the cells.
On the other hand, it was reported by F. A. M. Kalver and R. van der Meer in Appl. Environ. Microbiol. (1993), 59: 1120-1124 that Lactobacillus and Bifidobacterium could not assimilate cholesterol, but were able to reduce cholesterol contents in a medium by increasing the conjugating activity of bacterial bile salts when the pH value was below 6.0 to result in co-precipitation of cholesterol with bile salts (this phenomenon may be related to the BSH activity of the bacteria).
In 1997, M. M. Brashears and S. E. Gililland indicated that Lactobacillus exhibited good cholesterol co-precipitation during growth without pH control (i.e., at a normal pH of 4.5 to 5.5). However, if the pH value was maintained at about 6.0, the ability of said bacteria to remove cholesterol is significantly decreased (M. M. Brashears and S. E. Gililland (1997), Influences of pH during growth on removal of cholesterol from MRS broth by Lactobacillus casei and Lactobacillus acidophilus, Animal Science Research Report, pp. 32-37).
In 1998, Zhang Jia-cheng et al. tried to prove that “assimilation” is the major mechanism in the reduction of cholesterol by lactic acid bacteria by experimenting with high lipid milk and edible oil (Zhang Jia-cheng et al. (1998), “The research of cholesterol elimination in food by lactic acid bacteria—the screening of lactic acid bacteria species (strains),” Food Science (P.R.O.C), 19:20-22).
In 1999, Usman and Hosono indicated in J. Dairy Sci., 82: 243-248 that a newly found Lactobacillus species, Lactobacillus gasseri, can take up cholesterol under culture conditions without bile salts.
Using a biological method to reduce serum cholesterol levels in humans will be more economical and effective. It is apparent from the aforesaid references that lactic acid bacteria may exhibit the effect of reducing cholesterol both in vivo and in vitro, and the possible mechanisms include deconjugation by bile salt hydrase (BSH), co-precipitation of cholesterol with deconjugated bile salts under acidic conditions, and assimilation of cholesterol by lactic acid bacterial cells.
However, after being ingested into the human body, lactic acid bacteria encounter pressure from the gastrointestinal environment and specificity of intestinal absorption. Therefore, lactic acid bacteria have to first overcome the unfavorable environment of the digestive system and colonize the intestinal tract in order to grow and exert their reaction(s) in the intestinal tract. Further, Lactobacillus acidophilus strains are a group of bacteria having complex nutritional requirements. The bacteria are relatively stable in fermented milk. However, the bacteria in commercially available products (in forms of dry powder, grains, tablets) can hardly remain viable after long-term storage at room or refrigerated temperature so that the level of the bacteria cannot be easily maintained at an initial storage level. Accordingly, the actual number of bacteria in a non-fermented milk product is often less than that indicated on the product label. Hence, how to maintain the level of lactic acid bacteria in lactic acid bacteria products during marketing and storage is of utmost importance to manufacturers. Furthermore, screening bacteria to obtain bacterial strains having good resistance to acids and bile salts and capable of lowering serum cholesterol is an important subject in the development of excellent Lactobacillus products.
As mentioned above, if resistance to acids and bile salts and storage stability are taken into account when screening bacteria, cost savings can be achieved in the subsequent manufacturing process, and the screened bacterial strains can have a wider range of applications. In addition, the screening for effective bacterial strains is also related to origin and locality. Therefore, in recent years, investigators' efforts have focused on the screening of bacterial strains of human origin.
In Japan, in a total of 171 FOSHU (Foods for Specified Health Use)-approved products, 36 contain probiotic bacteria. They make up about 21% of the total number of products, but have a production value of up to 82%. In Europe, the production value of probiotic bacteria in the food market amounts to 1 billion US dollars. In the United States, yogurt sales in 2000 were up to 1.86 billion US dollars. In Taiwan, the probiotic bacteria market grew to NT$ 4.2 billion dollars in 2000. The applications of probiotic bacteria have increased every year and are no longer restricted to fermented milk, milk, ice cream, candies, and dietary supplements. The products are consumed by adults, infants, poultry, and livestock. It is anticipated that there is huge room for growth of the probiotic market.
There are a number of studies on lactic acid bacteria worldwide. Many of the patents and publications on the acid tolerance and cholesterol lowering ability of Lactobacillus sp. are directed to Lactobacillus acidophilus, and are largely concerned with bile- and acid-resistant bacterial strains, or are mainly focused on their ability to reduce cholesterol and bile tolerance. At present, studies carried out on the bile tolerance of Lactobacillus sp. emphasize that the bacterial strains can grow in an environment containing 0.3% glycocholate, and acid tolerance is tested using media at pH 2, the acidic condition occurring in the gastrointestinal tract at the initial stage of gastric juice secretion.
The ability of Lactobacillus sp. to reduce serum cholesterol is about 20% according to published patents, and is in the range from about 10% to about 80%, depending on the methods used, according to publications.
U.S. Pat. Nos. 4,839,281 and 5,032,399 disclose a strain, L. acidophilus GG (ATCC 53103), which was isolated from human feces. This strain can grow in an environment containing 0.15% bile salts, and the amount of residual bacteria after being cultured for 2 hours at pH 1-2 is 103 CFU.
It was reported by S. E. Gilliland and D. K. Walker in J. Dairy Sci.: 73:905-911 (1990) that L. acidophilus ATCC 43121 (corresponding to CCRC 17064) of pig origin and L. acidophilus ATCC 4356 (corresponding to CCRC 10695) of human origin can both grow in MRS broth supplemented with 0.3% bile salts and have the ability to reduce serum cholesterol.
Further, L. acidophilus LA16, the strain isolated from pigs as reported by Danielson et al. in J. Anim. Sci. (1989), 67:966-974, and Lactobacillus acidophilus DDS-1, an isolated strain of human origin (an endogenous human strain), which was developed by scientists headed by Dr. Khem M. Shahani and which is now a patented product of Nebraska Cultures, Inc., have demonstrated the ability to reduce serum cholesterol.
Usman and Hosono reported in J. Dairy Sci., 82: 243-248 (1999) that an isolated Lactobacillus strain Lactobacillus gasseri exhibited resistance to acids and bile salts and the ability to reduce cholesterol.
In U.S. Pat. Nos. 5,516,684 and 5,707,854, Yoshio Saito and Jun Mizutani disclose two strains of L. acidophilus, namely, L. acidophilus FERM-P-14204 and L. acidophilus FERM-P-14205. These strains do not exhibit deconjugation of bile acids and do not inhibit nutrient absorption, but they demonstrate the lowering of cholesterol in blood and liver.
However, the aforesaid Lactobacillus strains are of foreign origin. It would be desirable to obtain Lactobacillus isolates which are acid- and bile-resistant and which are capable of lowering serum cholesterol by screening bacteria strains in Taiwan so that the isolates thus obtained can adapt to the gastrointestinal environment of the people of Taiwan and, when used as starters or when added to processed products, can reach the intestine and colonize the intestine after ingestion to thereby enhance the functionality of the products.